Histone post-translational modifications (PTMs) have been intensively investigated due to their essential function in eukaryotic genome regulation. Histone modifications have been effectively studied using modified bottom-up proteomics approaches; however, the methods often do not capture single-molecule combinations of PTMs (proteoforms) that mediate known and expected biochemical mechanisms. Both middle-down mass spectrometry (MS) and top-down MS quantitation of H4 proteoforms present viable access to this important information. Histone H4 middle-down has previously avoided GluC digestion due to complex digestion products and interferences; however, the common AspN digestion cleaves at amino acid 23, disconnecting K31ac from other PTMs. Here, we demonstrate the effective use of GluC-based middle-down quantitation and compare it to top-down-based quantitation of proteoforms. Despite potential interferences in the m/z space, the proteoforms arising from all three GluC products (E52, E53, and E63) and intact H4 are chromatographically resolved and successfully analyzed in a single LC-MS analysis. Quantitative results and associated analytical metrics are compared between the different analytes of a single sample digested to different extents to reveal general concordance as well as the relative biases and complementarity of each approach. There is moderate proteoform discordance between digestion products (e.g., E52 and E53); however, each digestion product exhibits high concordance, regardless of digestion time. Under the conditions used, the GluC products are better chromatographically resolved yet show greater variance than the top-down quantitation that are more extensively sampled for MS2. GluC-based middle-down of H4 is thus viable. Both top-down and middle-down approaches have comparable quantitation capacity and are complementary.

中文翻译：

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GluC消化的组蛋白H4的一锅法上下定量分析。

由于组蛋白的翻译后修饰（PTM）在真核基因组调控中的重要功能，因此已经进行了深入研究。使用改良的自下而上的蛋白质组学方法已经有效地研究了组蛋白修饰。但是，这些方法通常无法捕获介导已知和预期生化机制的PTM（蛋白形式）的单分子组合。H4蛋白形式的中下质谱（MS）和自上而下的MS定量均提供了获取此重要信息的可行途径。由于复杂的消化产物和干扰，组蛋白H4中下水平以前避免了GluC消化。但是，常见的AspN消化在23位氨基酸处裂解，使K31ac与其他PTM断开连接。这里，我们证明了基于GluC的中下定量的有效利用，并将其与基于蛋白质的自上而下的定量进行了比较。尽管在m / z空间中存在潜在的干扰，所有三种GluC产物（E52，E53和E63）和完整的H4产生的蛋白形式都可以通过色谱分离，并在一次LC-MS分析中成功进行了分析。将定量分析的单个样品的不同分析物之间的定量结果和相关的分析指标进行不同程度的比较，以揭示总体一致性以及每种方法的相对偏差和互补性。消化产物（例如E52和E53）之间存在适度的蛋白形式不一致。但是，无论消化时间长短，每种消化产物都具有很高的一致性。在所使用的条件下，与对MS2进行更广泛采样的自上而下的定量分析相比，GluC产品的色谱分离效果更好，但显示出更大的方差。因此，基于GluC的H4中间态是可行的。自上而下和中下方法都具有可比的定量能力，并且是互补的。